Static separation method using non-porous cellulose beads

ABSTRACT

An affinity separation method and system comprising an affinity separation media with low porosity and low non-specific binding, and a fluid containing a target compound to be isolated which is capable of binding onto the affinity separation media in a fluid mixing loop in a static filtration apparatus. The static filtration apparatus comprises an intermixing-chamber containing a filtration medium wherein a tangential flow is created for intermixing the affinity separation media and target compound in the fluid. The fluid is capable of passing through the filtration median while the affinity separation media are substantially incapable of passing through the filtration medium. The affinity separation media are separated from the fluid by opening the filtrate outlet so as to allow the fluid to pass through the filtration medium of the static filtration apparatus. The filtrate can be thereby rendering substantially free of the target compound.

RELATED APPLICATION

[0001] The present application claims the benefit of the filing datesunder 35 U.S.C. § 119(e) to provisional U.S. patent application Ser. No.60/087,902 filed on Jun. 2, 1999, which is hereby incorporated byreference.

BACKGROUND OF THE INVENTION

[0002] Purification systems using glass or metal tubes which contain apacked column of separation medium, for example, beads or particles, areknown. These tubes are known as column boxes. Because the separationmedium is compacted within the column boxes, the flow rates are slow andthe column boxes have a limited capacity. Therefore, prior artpurification technology has focused on increasing the porosity of theseparation medium to increase the flow rates and capacity within thecolumn box. The object of these known systems is to purify the largestamount of material within the shortest amount of time while keeping theamount of contaminants low and the product yields high. One problem withthe old purification technology is that increasing porosity of theseparation medium achieved faster flow rates and capacity, but reducedproduct yields and quality.

[0003] A past method, which does not use a column box, utilized adynamic (i.e., mechanical forces at the site of filtration) filtrationsystem with a variety of separation medium. This old method andapparatus also failed in part because the sheering forces of the dynamicsystem damaged the separation medium and the bio-compounds beingpurified.

[0004] Prior methods of affinity separation involving dynamic filtrationof cellulose and non-cellulose beads have the disadvantage that thebeads degrade due to sheering forces inherent in dynamic filtration.These sheering forces irreversibly damage bead supports and thesheer-sensitive biological molecules grafted or bound to them.Generally, many porous beads will fragment due to the agitation andsheering forces contained in the dynamic affinity separation apparatus.The present invention overcomes the disadvantages of the prior art byutilizing a static affinity separation method which is gentle on beadsand biological molecules, but causes intermixing of the target compoundto be purified with the sheer-sensitive beads and biological moleculeswhich flow through the static tangential flow filters.

[0005] The prior art discloses the use of polystyrene beads in dynamicfiltration apparatuses for affinity separation of biological compounds.Certain disadvantages are involved in the use of polystyrene beads, suchas high non-specific adsorbing of biological molecules on theirhydrophobic surfaces. Further, polystyrene beads have open pores on thesurface of the bead which entrap contaminants which will co-purify withwanted products and decrease yield and purity of a target compound.Polystyrene has been found to exhibit a high degree of agglomerate andto adhere to the filters used in dynamic affinity separation methods.This agglomeration of the polystyrene beads allows for debris to becometrapped and spoils the filtration affinity system by clogging thefilter. A preferred embodiment of the present invention overcomes thedisadvantages of the prior art by adding a multitude of linkers to thebead surface to increase bead coating. Bead coating reduces unwantedagglomeration and filter clogging. This multitude of linkers reducesagglomeration and non-specific binding, resulting in increased stabilityand reduced entrapment of unwanted contaminants, while enabling theattachment of the target compounds desired to be separated.

[0006] The prior art discloses the use of cellulose in dynamicfiltration apparatuses for affinity separation of biological compounds.The prior art cellulose particles available are highly porous particleswhich exhibit entrapment of target and contaminants. Moreover, thesehighly porous prior art cellulose particles can swell from 25% to 400%of their original size in aqueous medium. Additionally, the prior artcellulose particles are highly sensitive to sheering forces, thusresulting in fragmentation in a dynamic filter. The present inventionuses non-porous cellulose beads in a static tangential flow system toprevent bead fragmentation.

BRIEF SUMMARY OF THE INVENTION

[0007] A preferred embodiment of the present invention provides animproved affinity separation method and system comprising an affinityseparation media with low porosity and low non-specific binding, and afluid containing a target compound to be isolated which is capable ofbinding onto the affinity separation media in a fluid mixing loop in astatic filtration apparatus. The static filtration apparatus comprisesan intermixing-chamber containing a filtration medium having upstreamand downstream sides, an inlet in fluid communication with the upstreamside of the filtration medium, and a filtrate outlet in fluidcommunication with the downstream side of the filtration medium, whereina tangential flow is created for intermixing the affinity separationmedia and target compound in the fluid. The fluid is capable of passingthrough the filtration medium while the affinity separation media aresubstantially incapable of passing through the filtration medium. Theaffinity separation media are separated from the fluid by opening thefiltrate outlet so as to allow the fluid to pass through the filtrationmedium of the static filtration apparatus. The filtrate can be therebyrendered substantially free of the target compound. If the recovery ofthe target compound is desired and/or if the affinity separation mediaare to be reused, the affinity separation media are then thoroughlywashed, and the target compound is eluted from the affinity separationmedia, filtered, and ultimately the target compound is recovered fromthe filtrate and the affinity separation media may be reused.

[0008] The preferred invention apparatus and system ideally utilizessmall, reconstituted cellulose particles having no pores and lownon-specific binding properties. The cellulose particles of thepreferred embodiment of the present invention consist of small,substantially spherical bodies with a near complete absence ofirregularities, holes, cracks and the like. The cellulose bodies aremade from viscose. This improvements results in uncross-linked, highdensity, spherical cellulose separation support beads withoutsubstantial holes, voids or craters on their surface. In certaincircumstances, as where ligands are attached to the cellulose particles,chromatographic separation may be optimized when substrate/sorbentinteractions take place exclusively on the outside surface of theparticle. In such cases, any presence of holes of a size that mayaccommodate a substrate molecule cannot be tolerated; otherwisediffusion based interferences may adversely effect resolution of purecompounds.

[0009] The cellulose beads are essentially non-crystalline. Electronmicrograph sections of the particles mounted in an epoxy matrix displaya structure whereby the cellulose particles show a dense non-porousouter shell with an approximate thickness of 1,000 to 2,000 angstromsand a more porous interior of the closed-cell type. The shape of theparticles is essentially spherical. The cellulose particles areessentially non-swellable and stable in pH range between about 1 and 13.

[0010] The current invention relates to a unique and novel method whicheliminates the column box and utilizes low porosity. The present methodfor the affinity separation of bio-compounds uses a static filtrationsystem instead of the column box to achieve flow rates of liters perminute instead of milliliters per minute, which is typical of highporosity separation media. Therefore, a low porosity, low non-specificbinding separation medium, generally the lowest being reconstitutedcellulose affinity particles, is preferred.

[0011] An advantage of the present inventive method is the provision ofthe exceptionally efficient separation through an affinity separationprocedure of a compound from a dilute solution. A preferred embodimentof the present invention provides a means for lessening the number ofprocessing steps required to perform an affinity separation as comparedto known affinity separation methods, thereby increasing the overallyield of the separation method.

[0012] A further advantage of the preferred embodiment of the inventionis that the present inventive method is able to be conducted in arelatively lesser amount of time as compared to known affinityseparation processes.

[0013] Moreover, a further advantage of the invention is that since thepresent inventive method preferably utilizes nonporous affinityparticles, the present invention avoids those problems attendant the useof highly porous affinity particles, e.g., fragility during mechanicalagitation, affinity particle fouling, susceptibility to crushing, andswelling.

[0014] In addition, it is yet another advantage of the invention thatthe present inventive method generally avoids problems associated withchanneling and filtration medium fouling associated with conventionalchromatographic affinity separation methods using column boxes.

[0015] An embodiment of the present invention overcomes thedisadvantages of the prior art by adding a multitude of linkers to thebead surface to coat the bead. Thereby, the bead pores are covered, thusreducing unwanted porosity and non-specific binding which may allow forentrapment and adsorption of unwanted contaminants. The linkers may beadded to the beads by covalent bonding or in other manners known tothose skilled in the art. This linker coating enables the attachment ofspecial chemical “hooks” which specifically bind the desired targetcompounds to be separated.

[0016] A further advantage of a preferred embodiment of the presentinvention over the prior art is that the use of reconstituted celluloseparticles prevents the entrapment of target molecules and contaminantsand the swelling associated with the use of prior art cellulose beads.In comparison, a reconstituted cellulose particle swells no more than15% of its original size.

[0017] A further advantage of a preferred embodiment of the presentinvention over the prior art is that static tangential affinity systemsprevent the accumulation of debris and beads that usually connect andclog tangential flow systems that may get trapped in systems such as theprior art dynamic systems.

DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 is a schematic depiction of the significant elements usedin a preferred embodiment of the present invention.

[0019]FIG. 2 is a schematic depiction of a preferred embodiment of theapparatus of the present invention for use with the low porosity, lownon-specific binding separation media, the preferred separation mediabeing reconstituted cellulose particles.

[0020]FIG. 3 is a schematic depiction of an embodiment of the presentinvention for sequential purification of biomaterials.

DETAILED DESCRIPTION OF THE DRAWINGS AND PREFERRED EMBODIMENTS OF THEINVENTION

[0021] The present invention provides an improved affinity separationmethod. It has been found that the use of a static filtration apparatushaving a tangential flow can be used to efficiently effect separationbetween a fluid without plugging the filter and suspended thereinaffinity particles. The static filtration apparatus can thereafter beused to separate the fluid from the nonporous cellulose affinityparticles having the bound or complexed target thereon. Such a processenables the use of nonporous cellulose affinity particles having smalldiameters and relatively large surface areas per weight of thecellulose.

[0022] The preferred inventive method is an improved affinity separationmethod comprising:

[0023] (a) introducing reconstituted cellulose affinity particles into afluid containing a target compound to be isolated which is capable ofspecifically binding onto the cellulose affinity particles by means ofspecific “hooks” which specifically bind the target compound,

[0024] (b) filtering the resulting suspension in a static filtrationapparatus, comprising a filtration medium having upstream and downstreamsides, an inlet in fluid communication with the upstream side of thefiltration medium, and a filtrate outlet in fluid communication with thedownstream side of the filtration medium, wherein the apparatus has atangential flow and the fluid is capable of passing through thefiltration medium and the nonporous cellulose affinity particles aresubstantially incapable of passing through the filtration medium, and

[0025] (c) separating the cellulose affinity particles from the fluid byopening the filtrate outlet so as to allow the fluid to pass through thefiltration medium of the static filtration apparatus.

[0026] As a consequence of affinity of the target compound for the beadsurface, which has been rendered attractive to the target compound, thetarget concentration in the fluid can be brought down to sufficientlylow levels, allowing such process to be used as a practical industrialmethod of separation of individual compounds from a mixture of amultitude of compounds in a solution.

[0027] The relative sizes of the largest debris in the fluid to betreated, the pores of the filtration medium, and the diameter of thereconstituted affinity particles are important in the practicalutilization of the present inventive affinity separation method.Ideally, the filtration medium will allow all of the fluid (includingthe largest debris) to pass therethrough, while preventing any of thereconstituted cellulose affinity particles from passing therethrough.The filtration medium, therefore, will preferably have an average poresize at least a factor of two, and preferably a factor of five or ten,larger than the largest debris in the fluid to be treated, and thereconstituted cellulose affinity particles will preferably be at least afactor of two, and preferably a factor of five or ten, larger than thepore size of the filtration medium. Thus, for example, if the largestdebris in the fluid to be treated is in on the order of about 0.1-0.2micron, the average pore size of the filtration medium will preferablybe on the order of about 1-3 microns, while the reconstituted celluloseaffinity particles will preferably be on the order of about 10-20microns.

[0028] In general, the smallest acceptable reconstituted celluloseaffinity particles, e.g., reconstituted cellulose affinity particles of60 microns diameter or less, particularly reconstituted celluloseaffinity particles of 20 microns diameter or less, are preferably usedto improve the recovery of the target compound by increasing theavailable surface area per unit of weight of the reconstituted celluloseaffinity particles for interaction with the fluid being treated. Therange of size for the reconstituted cellulose affinity particles canrange from nano-size to 120 microns. Accordingly, while pretreatment ofthe fluid to be treated may not be the most desirable inasmuch as itadversely impacts on the recovery of the target compound by introducingan additional processing step, there may be instances in which therecovery loss resulting from pretreatment, particularly prefilteringand/or homogenization to reduce the sizes of the largest debris in thefluid, will be more than offset by the resulting ability to use smalleraffinity particles having a higher area per unit of weight of theaffinity particles.

[0029] Although several separation media may be modified to yieldaffinity beads which possess low porosity and reduced nonspecificbinding, the preferred embodiment includes nonporous cellulose beadswith reduced nonspecific binding. The reconstituted cellulose affinityparticles comprise a spacer and a ligand on the surface thereof which iscapable of binding to the target compound in a fluid so as to enable theseparation of that compound from the remainder of the fluid. Then, thetarget compound is capable of being removed from the reconstitutedcellulose affinity particles by changing the conditions of the solution,for example, pH, salts, and others. The affinity ligands used toseparate particular compounds from a fluid will vary. The properselection of the ligand will ensure that the target compound selectivelyand reversibly binds, e.g., complexes with or adsorbs onto, thereconstituted cellulose affinity particle.

[0030] In a preferred embodiment, low porosity, low non-specific-bindingseparation media includes low porosity linker coated polystyrenecarbohydrate, glass, porcelain, ceramics, and other low porosity beadsknown to one skilled in the art. Examples of beads which may be used inthe present invention are disclosed in U.S. Pat. No. 5,567,615 to Deganet al. the entire disclosure of which is incorporated herein byreference. In yet another preferred embodiment, the separation mediumincludes reconstituted, nonporous cellulose particles. In thisembodiment, reconstituted cellulose particles exhibit pore-free surfaceswith linkage and functional groups in the bulk of the solution free toform attachments to the target compounds that are desired to beseparated. The reconstituted cellulose particles have no pores whichopen onto the particle surface into which may be trapped a targetcompound to be separated, thereby lowering yield. Further, celluloseparticles can be constructed to be of consistent, small size whichprovides for increase in surface area of contact and a uniform flow inthe affinity separation apparatus.

[0031] Fusion proteins, where a target protein or a peptide is fusedwith segments such as glutathion transferase or polyhystidine, requiregrafted affinity ligands, glutathion, or chelated metals, such as Nickelor Zinc, respectively. In an embodiment of the invention, affinityseparation of fusion proteins uses ungrafted cellulose beads of thepresent invention, without spacers or ligands, for separation of fusionproteins comprising a cellulose binding protein segment fused with atarget protein or peptide. In this particular case, the cellulosesurface of the beads as a whole represent an affinity site for attachingsaid cellulose binding protein segment.

[0032] The reconstituted cellulose affinity particles preferably have asurface which is smooth and nonporous and are nearly identical.

[0033] The reconstituted cellulose affinity particles are preferablysubstantially spherical shaped. Moreover, the reconstituted celluloseaffinity particles are preferably of narrow distribution in diameter.Such reconstituted cellulose affinity particles generally will have thelowest probability of creating flow problems and will allow an easyselection of membrane porosity for fast and clean separation.

[0034] The reconstituted cellulose affinity particles may be of anysuitable size. The average diameter of the reconstituted celluloseaffinity particles will typically be less than about 120 microns, andmore usually less than about 60 microns. Preferably, the affinityparticles will have an average diameter of less than about 20 microns,and more preferably less than about 10 microns. For many uses, thereconstituted cellulose affinity particles will advantageously have adiameter of about 1 to about 5 microns. The reconstituted celluloseaffinity particles preferably have a narrow diameter distribution.

[0035] The preferred particles of the present invention are of about 1-3microns in diameter. Such small particles have relatively high surfacearea per unit volume and are easily filtered in a static filterapparatus. The efficiency of the overall affinity separation method ofthe present invention is high because the near complete recovery of thereconstituted cellulose affinity particles with the bound targetcompound is achieved.

[0036] The surface of the preferred cellulose beads has low or noaffinity for the target compound in the fluid being treated. Such asurface ensures that the target compound will reversibly complex orotherwise attach exclusively to the ligand on the surface of theparticles rather than the surface of the particles itself, where othercomponents or compounds as well might get nonspecifically bound, therebycausing impurities to be present with the target compound. Moreover, thesurface of the substrate is preferably smooth to minimize adherence ofmaterial other than the target compound to the attached ligand on thesurface of the reconstituted cellulose affinity particles and to providefor easy cleaning of the reconstituted cellulose affinity particles. Thebeads are preferably capable of being reused many times and arechemically and mechanically stable such that the beads do not decomposeor otherwise pose contamination problems.

[0037] There are potentially endless numbers of ligands for affinityseparation of a given target compound. To mention a few generallyapplicable examples, antibodies of the monoclonal and polyclonal kind,active site analogs, chelated metals, particularly Nickel, Zinc, andCopper are used in the present art as affinity ligands.

[0038] The filtration medium used in conjunction with the staticfiltration apparatus can be any suitable filtration medium and willtypically be a porous membrane, preferably a microporous polymericmembrane. While the filtration medium may have any suitable pore rating,e.g., on average of about 20 microns or less, on average of about 10microns or less, on average of about 5 microns or less, or even about onaverage of 1 micron or less, the filtration medium will preferably allowfor all, or at least substantially all, of the fluid being treated topass through the filtration medium while retraining all or at leastsubstantially all, of the reconstituted cellulose affinity particles.Thus, the pore rating of the filtration medium is largely dependent onthe size of the reconstituted cellulose affinity particles and the sizeof the largest debris (i.e., nontarget compound and/or particulate) inthe fluid being treated.

[0039]FIG. 2 schematically depicts a preferred static filtrationapparatus for use with the present inventive affinity separation method.The static filtration apparatus comprises a housing 20 having a feedport 21, a concentrate port 22, and a filtrate port 23. A filtrationmedium or membrane 25 is disposed within the housing 20. The feed port21 and concentrate port 22 are in fluid communication with the upstreamside of the filtration medium 25, while the filtrate port 23 is in fluidcommunication with the downstream side of the filtration medium 25.Fluid is capable of passing through the filtration medium 25, while thereconstituted cellulose affinity particles are substantially incapableof passing through the filtration medium 25.

[0040] In use, a mixture of the fluid containing the target compound andthe reconstituted cellulose affinity particles are fed into the housing20 of the static filtration apparatus via feed port 21 in such a manneras to produce a tangential flow.

[0041] The mixture is then filtered through filtration medium 25 byopening filtrate port 23 to allow the fluid to be removed from withinthe static filtration apparatus. Washing buffer may be introduced intothe static filtration apparatus via feed port 21 to remove any unboundmaterial within the static filtration apparatus, and the washing buffermay then be allowed to pass through the filtrate port 23. Multiplewashing steps, if desire, can be carried out in the same manner.

[0042] Eluent is then introduced, via feed port 21, to detach the targetcompounds from the reconstituted cellulose affinity particles. After thetarget compound has been removed from the reconstituted celluloseaffinity particles and recovered, the reconstituted cellulose affinityparticles can then be reused after washing. The washing buffer isremoved by way of port 23.

[0043]FIG. 2 is merely a schematic illustration of a preferredembodiment of the apparatus useful in the carrying out of the presentinventive method, and particular aspects of an actual apparatus for usewith the present invention may vary considerably.

EXAMPLE 1

[0044] The present invention may be further understood with reference tothe accompanying drawings. FIG. 1 schematically depicts the significantelements used in a preferred embodiment of the present invention. Uponcommencement of the present inventive methods, the fluid to be treated,which contains the target compound to be separated from the remainder ofthe fluid, resides in holding tank 1, while the affinity particlesreside in holding tank 2. Both the fluid and the affinity particles aretransferred to a buffer tank 3 where they are combined to form amixture. The mixture is then transferred to the static filtrationapparatus 4 via valve 14 and inlet 5, although the fluid andreconstituted cellulose affinity particles could be directly transferredto the static filtration apparatus 4 without passing through the buffertank 3. The mixture is maintained in the static filtration apparatus 4without any of the fluid passing through the filtration medium of thestatic filtration apparatus 4, by, for example, the filtrate valve 7being in the closed position. Intermixing of the fluid and thereconstituted cellulose affinity particles is provided by an optionalagitator 12 or by a tangential fluid in tank 3.

[0045] Thereafter, some or all of the mixture is transferred viaconcentrate valve 8 in a batch or continuous (in-line) process to adetection tank 9, wherein the concentration of the target compound inthe fluid which remains unbound to the reconstituted cellulose affinityparticles is determined. In a permanent manufacturing process whereinsimilar batches of fluid are being repeatably treated, there may be noneed for any detection means after determining an appropriate quantityof fluid, reconstituted cellulose affinity particles, and residence timein the static filtration apparatus inasmuch as the present inventivemethod is quite consistent and reproducible as regards the recovery ofthe target compound.

[0046] When it is determined, by whatever means, that a sufficientamount of target compound has been adsorbed onto the reconstitutedcellulose affinity particles, then the filtrate valve 7 is opened sothat the fluid passes through the filtration medium of the staticfiltration apparatus 4 into waste tank 6, thereby leaving thereconstituted cellulose affinity particles in the static filtrationapparatus 4. The filtration valve 7 is then typically closed, although,alternatively, additional fluid can be passed into the static filtrationapparatus 4 for contacting with the reconstituted cellulose affinityparticles, particularly if the reconstituted cellulose affinityparticles are not saturated with the target compound. Such additionalfluid can be passed into the static filtration apparatus 4 in acontinuous or semi-continuous manner while some of the fluid in thestatic filtration apparatus 4 continues to pass through the filtrationmedium of the static filtration apparatus 4.

[0047] After the addition of fluid into the static filtration apparatus4 is complete and the fluid within the static filtration apparatus 4 haspassed through the filtration medium, the washing buffer valve 11 isopened to allow for washing buffer from washing buffer tank 10 to enterthe static filtration apparatus 4. The washing buffer is allowed tointermingle with the reconstituted cellulose affinity particles for asuitable period of time, and then the filtrate valve 7 is again openedto allow for the washing buffer to pass to the waste tank 6. Typically,there will be several such wash cycles to ensure that the fluid beingtreated, except for the target compound bound to the reconstitutedcellulose affinity particles, has been removed from the staticfiltration apparatus 4.

[0048] The reconstituted cellulose affinity particles are thentransferred to the elution receptacle 13 via the concentrate valve 8.Such a transfer may be accomplished by using washing buffer from thewashing buffer tank 10 to transport the reconstituted cellulose affinityparticles to elution receptacle 13. Eluent from eluent tank 15 thencontacts the affinity particles in the receptacle 13 to detach thetarget compound from the reconstituted cellulose affinity particles andpasses the target compound, together with beads, back into the staticfilter 4 via valve 14 and inlet 5. The effluent stream exits the staticfilter via valve 7 and is diverted into tank 7. An optional effluentdetection means which monitors the level of a target compound in theeffluent stream may also be installed, similar to detection tank 9.

[0049] The reconstituted cellulose affinity particles, which no longerhave the target compound bound thereto, can consequently be washed andthen reused. After washing with buffer from tank 10, regenerated beadsare transferred back to tank 2 via concentrate valve 8.

[0050]FIG. 1 is merely a schematic illustration of a preferredembodiment of the present invention, and the actual equipment and itsplacement may be varied considerably for the embodiments of the presentinvention.

EXAMPLE 2

[0051] As shown in FIG. 3, Example 2 comprises a series of purificationloops, each of which is a self contained purification system for aparticular target, e.g., a biomaterial. The purification loops areconnected, enabling the fluid, or permeate of the previous system to bethe “feed stock” or fluid to be treated, for the next loop. Each loopmay contain affinity particles possessing different hooks or the samehooks as the previous purification loop in the connected configuration.The loops begin with a feed tank 1′ which may be similar to the holdingtank 1 of FIG. 1. Each loop of affinity particle may purify a specificproduct from a single source of fluid to be treated which comprises amixture of products. For example, commonly human immunoglobulin, humanalbumen, and human clotting factors are purified from raw human plasmaby separate methods. Some of these methods in some cases damage the rawhuman plasma, thus preventing additional product extraction. The currentinvention describes a method which enables extraction of two or morevaluable bio-products from human plasma without damaging thebiomaterials within the used raw human plasma. FIG. 3 shows thesequential tangential flow systems where the first system purifiesbiomaterial x, the second biomaterial y, the third biomaterial z, and soon. X , y and z may represent the same or different biomaterials to bepurified. A solution containing all biomaterials passes through eachsystem containing bead loops which circulate low porosity particleshaving chemical hooks which specifically bind either x, y, or z, and soon. After the solution has passed through all of the beads loops and x,y, and z biomaterials have been removed within each of the specific beadloops, the bead loops are disconnected from each other and each loopsystem is separately processed to yield purified biomaterials x, y, andz.

[0052] It should be appreciated that the method and system of thepresent invention is capable of being incorporated in the form of avariety of embodiments, only a few of which have been illustrated anddescribed above. The invention may be embodied in other forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive and the scope of the invention is, therefore,indicated by the appended claims rather than by the foregoingdescription. All changes that come within the meaning and range ofequivalency of the claims are to be embraced within their scope.

We claim:
 1. A method of separation for isolating a target compound froma fluid comprising: (a) forming a suspension comprising i) a fluidcontaining a target compound to be isolated, and ii) a plurality of lowporosity affinity particles capable of binding the target compound, (b)maintaining said low porosity affinity particles and said fluid incontact for a sufficient period of time for binding of said targetcompound onto said low porosity affinity particles to be effected, (c)introducing said suspension into a static filtration apparatus having afiltration medium, (d) creating a tangential flow in said staticapparatus thereby aiding in separation between said fluid and said lowporosity affinity particles with the target compound attached to thesurface thereof, (e) passing said fluid devoid of the target compoundthrough said filtration medium of said static filtration apparatus, (f)washing said low porosity affinity particles, and (g) eluting saidtarget compound from said low porosity affinity particles.
 2. The methodof claim 1, wherein said low porosity affinity particles are nonporous.3. The method of claim 2, wherein said low porosity affinity particlesare generally spherical.
 4. The method of claim 3, wherein said lowporosity affinity particles are generally identical in diameter.
 5. Themethod of claim 4, wherein said low porosity affinity particles have anaverage diameter of between about 0.5 and about 25 microns.
 6. Themethod of claim 4, wherein said low porosity affinity particles have anaverage diameter between about 1 and about 3 microns.
 7. The method ofclaim 4, wherein said low porosity affinity particles have an averagediameter at least about two-fold larger than the average pore size ofsaid filtration medium.
 8. The method of claim 7, wherein said fluidcontains particulate and said filtration medium has an average pore sizeat least about two-fold larger than the size of the largest particulatein said fluid.
 9. The method of claim 4, wherein said low porosityaffinity particles have an average diameter at least about five-foldlarger than the average pore size of said filtration medium.
 10. Themethod of claim 7, wherein said fluid contains particulate and saidfiltration medium has an average pore size at least about five-foldlarger than the size of the largest particulate in said fluid.
 11. Themethod of claim 1, wherein said target compound is eluted from said lowporosity affinity particles by transferring said affinity particleshaving said target compound thereto from said static filtration to atank, passing an eluent through said tank to remove said target compoundfrom said low porosity affinity particles, thereby providing eluded lowporosity affinity particles from which said target compound has beenremoved, and collecting the resulting eluent containing said targetcompound.
 12. The method of claim 10, wherein said eluted low porosityaffinity particles are then transferred back to said static filtrationapparatus and the method is repeated.
 13. The method of claim 1, whereinsaid low porosity affinity particles have an average diameter betweenabout 0.5 and about 120 um.
 14. The method of claim 1, wherein said lowporosity affinity particles have an average diameter range of about 60urn or less.
 15. The method of claim 12, wherein said low porosityaffinity particles have an average diameter of about 20 um or less. 16.The method of claim 13, wherein said filtration medium has an averagepore rating of less than about 5 um.
 17. The method of claim 1, whereinsaid low porosity affinity particles have an average diameter of atleast about 0.5 um.
 18. The method of claim 15, wherein said filtrationmedium is a microporous membrane having an average pore rating of lessthan about 1 um.
 19. A system for affinity separation of a targetcompound from a fluid comprising: (a) a static filtration apparatuscomprising a housing having a feed port, a concentrate port, a filtrateport, and a filtration medium disposed within the housing; (b) the feedport and concentrate port being in fluid communication with the upstreamside of the filtration medium, while the filtrate port is in fluidcommunication with the downstream side of the filtration medium; and (c)a suspension comprising a plurality of low porosity affinity particlesand a fluid containing a target compound to be isolated fed into thefiltration apparatus via the feed port for inter-mixing.
 20. The systemof claim 19, wherein said low porosity affinity particles are nonporous.21. The system of claim 19, wherein said low porosity affinity particlesare generally spherical.
 22. The system of claim 19, wherein said lowporosity affinity particles are generally identical in diameter.
 23. Thesystem of claim 20, wherein said low porosity affinity particles have anaverage diameter at least about two-fold larger than the average poresize of said filtration medium.
 24. The system of claim 21, wherein saidfluid contains particulates and said filtration medium has an averagepore size at least about two-fold larger than the size of the largestparticulate in said fluid.
 25. The system of claim 20, wherein said lowporosity affinity particles have an average diameter at least aboutfive-fold larger than the average pore size of said filtration medium.26. The system of claim 23, wherein said fluid contains particulates andsaid filtration medium has an average pore size at least about five-foldlarger than the size of the largest particulate in said fluid.
 27. Thesystem of claim 19, wherein said low porosity affinity particles have anaverage diameter of about 120 um or less.
 28. The system of claim 19,wherein said low porosity affinity particles have an average diameter ofabout 60 um or less.
 29. The system of claim 19, wherein said lowporosity affinity particles have an average diameter or about 20 um orless.
 30. The system of claim 27, wherein said filtration medium has anaverage pore rating of less than about 5 um.
 31. The system of claim 19,wherein said low porosity affinity particles have an average diameter ofbetween about 1 and about 3 um.
 32. The system of claim 19, wherein saidfiltration medium is a microporous membrane having an average porerating of less than 1 um.
 33. The system of claim 19 wherein said lowporosity affinity particles are ungrafted for separation of a fusionprotein comprising a cellulose binding protein segment fused with atarget protein or peptide.
 34. The system of claim 33 wherein said lowporosity affinity particles comprise a bead having a surface, saidsurface of the bead as a whole representing an affinity site forattaching a cellulose binding fusion protein segment.
 35. The system ofclaim 19 wherein said low porosity affinity particles have a pluralityof chemical linkers attached thereto.
 36. The system of claim 19 whereinsaid low porosity affinity particles are linker-coated polystyreneaffinity particles having generally less than 40% porosity.
 37. Thesystem of claim 20 wherein said low porosity affinity particles arereconstituted cellulose affinity particles.
 38. The system of claim 19wherein said system is connected to other said systems forming amulti-system purification method wherein at least a product may bepurified using said low porosity affinity particles capable of bindingsaid products in each system for the purification of each productseparate from the other using a single source of raw material having amixture of each product.